Methods for the treatment of cellular proliferative disorders

ABSTRACT

The present invention relates to methods of identifying the susceptibility of cells to reovirus infection by measuring constitutive ras-MAP signaling. The invention also pertains to methods using reovirus for the treatment of cellular proliferative disorders, and particularly cellular proliferative disorders wherein the proliferating cells exhibit constitutive MAPK phosphorylation, in mammals. In particular, the methods provide for reovirus treatment of mammals to treat proliferative disorders which include breast tumors, a subset of tumors in which mutation of the ras gene is not believed to play a significant role.

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/246,728, filed Nov. 9, 2000, the disclosure ofwhich is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods of identifying thesusceptibility of cells to reovirus infection by measuring constitutiveras-MAP signaling. The invention also pertains to methods using reovirusfor the treatment of cellular proliferative disorders, and particularlycellular proliferative disorders wherein the proliferating cells exhibitconstitutive MAPK phosphorylation, in mammals. In particular, themethods provide for reovirus treatment of mammals to treat proliferativedisorders which include breast tumors, a subset of tumors in whichmutation of the ras gene is not believed to play a significant role.

[0003] References

[0004] The following publications, patent applications and patents arecited in this application:

[0005] U.S. Pat. No. 5,023,252.

[0006] WO 99/08692, published Feb. 25, 1999.

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[0009] Barbacid, M., Annu. Rev. Biochem., 56:779-827 (1987).

[0010] Baselga et al. (1996), J. Clin. Onc. 14:737-744.

[0011] Bos, J. (1989) Cancer Res. 49:4682.

[0012] Carter et al. (1992), PNAS 89:4285-4289.

[0013] Chandron and Nibert, “Protease cleavage of reovirus capsidprotein mul and mu1C is blocked by alkyl sulfate detergents, yielding anew type of infectious subvirion particle” J. of Virology 72(1):467-75(1998).

[0014] Chaubert, P. et al. (1994), Am. J. Path. 144:767.

[0015] Clark et al. (1995), Science 268:233-239.

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[0017] Cuff et al., “Enteric reovirus infection as a probe to studyimmunotoxicity of the gastrointestinal tract” Toxicological Sciences42(2):99-108 (1998)

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[0019] Duncan et al., “Conformational and functional analysis of theC-terminal globular head of the reovirus cell attachment protein”Virology 182(2):810-9 (1991).

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[0021] Fields, B. N. et al. (1996), Fundamental Virology, 3rd Edition,Lippincott-Raven.

[0022] Gentsch, J. R. K. and Pacitti, A. F. (1985), J. Virol. 56:356.

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[0027] Jacobs et al. (1983), Cancer Res 43:1696-1702.

[0028] Janes, P. W., et al. (1994)Oncogene 9:3601.

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[0036] Migliaccio et al. (1996), EMBO J 15:1292-1300.

[0037] Migliaccio et al. (1998), EMBO J 17:2008-2018.

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[0058] All of the above publications, patent applications and patentsare herein incorporated by reference in their entirety to the sameextent as if each individual publication, patent application or patentwas specifically and individually indicated to be incorporated byreference in its entirety.

[0059] State of the Art

[0060] Normal cell proliferation is regulated by a balance betweengrowth-promoting proto-oncogenes and growth-constrainingtumor-suppressor genes. Tumorigenesis can be caused by geneticalterations to the genome that result in the mutation of those cellularelements that govern the interpretation of cellular signals, such aspotentiation of proto-oncogene activity or inactivation of tumorsuppression. It is believed that the interpretation of these signalsultimately influences the growth and differentiation of a cell, and thatmisinterpretation of these signals can result in neoplastic growth(neoplasia).

[0061] Genetic alteration of the proto-oncogene Ras is believed tocontribute to approximately 30% of all human tumors (Wiessmuller, L. andWittinghofer, F. (1994), Cellular Signaling 6(3):247-267; Barbacid, M.(1987) A Rev. Biochem. 56,779-827). The role that Ras plays in thepathogenesis of human tumors is specific to the type of tumor.Activating mutations in Ras itself are found in most types of humanmalignancies, and are highly represented in pancreatic cancer (80%),sporadic colorectal carcinomas (40-50%), human lung adenocarcinomas(15-24%), thyroid tumors (50%) and myeloid leukemia (30%) (Millis, N Eet al. (1995) Cancer Res. 55:1444; Chaubert, P. et al. (1994), Am. J.Path. 144:767; Bos, J. (1989) Cancer Res. 49:4682). Ras activation isalso demonstrated by upstream mitogenic signaling elements, notably bytyrosine receptor kinases (RTKs). These upstream elements, if amplifiedor overexpressed, ultimately result in elevated Ras activity by thesignal transduction activity of Ras. Examples of this includeoverexpression of PDGFR in certain forms of glioblastomas, as well as inc-erbB-2/neu in breast cancer (Levitzki, A. (1994) Eur. J. Biochem. 226:1; Janes, P.W., et al. (1994) Oncogene 9:3601; Bos, J. (1989) CancerRes. 10 49:4682).

[0062] Current methods of treatment for neoplasia include surgery,chemotherapy and radiation. Surgery is typically used as the primarytreatment for early stages of cancer; however, many tumors cannot becompletely removed by surgical means. In addition, metastatic growth ofneoplasms may prevent complete cure of cancer by surgery. Chemotherapyinvolves administration of compounds having antitumor activity, such asalkylating agents, antimetabolites, and antitumor antibiotics. Theefficacy of chemotherapy is often limited by severe side effects,including nausea and vomiting, bone marrow depression, renal damage, andcentral nervous system depression. Radiation therapy relies on thegreater ability of normal cells, in contrast with neoplastic cells, torepair themselves after treatment with radiation. Radiotherapy cannot beused to treat many neoplasms, however, because of the sensitivity oftissue surrounding the tumor. In addition, certain tumors havedemonstrated resistance to radiotherapy and such may be dependent ononcogene or anti-oncogene status of the cell (Lee. J. M. et al. (1993)PNAS 90:5742-5746; Lowe. S. W. et al. (1994) Science, 266:807-810;Raybaud-Diogene. H. et al. (1997) J. Clin. Oncology, 15(3):1030-1038).

[0063] Breast cancer is amongst the most common and feared cancer inwomen with an estimated one in eight American women developing it intheir lifetime (NCI-SEER, 1998). Although there have been tremendousachievements in breast cancer treatment that have allowed a stablemortality rate in the face of an increasing incident rate, most of theseadvances can be attributed to improved early detection methods and notnovel treatment strategies. The fact that there have been few advancesin the actual treatment of this affliction demands that the developmentof unconventional treatment strategies be explored.

[0064] Never in the history of cancer biology has there been a morepromising period in the development of therapeutics for the treatment ofbreast cancer. Many of these therapeutics are targeted at specificreceptors that are overexpressed in subsets of this cancer, and are nowfinding some clinical use. Perhaps one of the most promising of thesenew therapies is the use of antibodies targeted to the HER2 receptorthat is frequently overexpressed in breast cancer. Results from the useof these monoclonal antibodies directed at the extracellular moieties ofthis growth factor receptor are promising, and indeed have met with someclinical success (Baselga, 1996; for reviews see Nass, 1998; Hung,1995). There are however a number of technical obstacles that must beaddressed. Firstly, these monoclonal antibodies directed against HER2are not themselves cytocidal. but are rather cytostatic (Pietras, 1994;Harwerth, 1992; Hudziak, 1989; Carter, 1992). Importantly, in a SCID ornude mouse model, anti-HER2 antibodies are effective only in repressingtumor growth and not actually causing tumor regression. Further, thisrepression of tumor growth is alleviated rapidly once the antibodyadministration is ceased. On a more positive note, there is evidencethat treatment of tumors with anti-HER2 antibodies in an immunecompetent animal may actually result in a cytocidal effect mediated byantibody-dependent cellular cytotoxicity (ADCC) (Carter, 1992).Secondly, targeting growth factor receptor (EGFR) may be of limitedvalue, as not all breast cancers express these receptors at elevatedlevels. Additionally, this strategy targets only receptor tyrosinekinases and would have little therapeutic benefit for those breastcancers that overexpress non-receptor tyrosine kinases such as Src,which have already been implicated in the growth of some breast cancers(Clark, 1996). Finally, the biology of the tumor blood vessels must alsobe taken into account as far as the capacities to deliver these largemacromolecules to their target cells. It has been demonstrated that thevasculature of a tumor is heterogeneous and the ability of a vessel toleak large macromolecules, such as antibodies, is a function of theirspatial orientation to the tumor mass (Dvorak, 1988; Nagy and Dvorak,1989). It has been shown that the vessels with the greatest permeabilityto these molecules reside primarily at the tumor-host interface, and theleast permeable vessels are those that actually penetrate the tumourmass (Dvorak, 1988). The result of this differential permeability isthat these tumour specific antibodies, and their cytocidal effectorcells, would fail to penetrate into the tumour mass and would be onlyeffective on the periphery of the mass.

[0065] Despite the fact that activating mutations in ras are infrequentin breast cancer, there is a growing body of evidence that activation ofthe Ras/MAPK pathway is important in the initiation and progression ofthis disease. Upstream elements of the Ras pathway, notably receptortyrosine kinases (RTK) are frequently overexpressed in breast cancers.HER-2/neu (erbB-2) is overexpressed in approximately 30% of all breastcancers (Spandidos 1987; Zhou, 1989; Archer, 1995) and is associatedwith poor patient prognosis (Slamon, 1989). When overexpressed inNIH-3T3 cells, HER-2 mediates transformation; however, there does appearto be a threshold level of overexpression that is required for thistransformation to occur (Jardines, 1993; Clark 1995). It appears thatthis transforming ability of HER-2 is dependent upon Ras activity ascell lines overexpressing HER-2 exhibit a dramatic increase in MAPkinase activity, the latter being reflective of Ras activity (Janes,1994). The growth factor receptor EGFR that is closely related to, butdistinct from HER-2, has also been observed to be overexpressed inbreast tumors and has also been correlated strongly with a poor patientprognosis (Shackney, 1998: Koenders, 1991).

[0066] Additional elements upstream of Ras have also been implicated inthe etiology of breast cancer. The non-receptor tyrosine kinase c-Srchas been implicated as being as important candidate in promoting theprogression of breast cancer. A number of studies indicate a −4 to −30fold increase of c-Src activity in primary breast cancer tumors whencompared to normal breast tissue (Verbeeks, 1996; Jacobs and Rubsamen,1983). C-Src has been suggested to also play an important role in thetransmission of signals from both the estrogen and progestin receptorsvia the Ras/MAPK pathway. A number of groups have observed that thetreatment of breast tumor derived MCF-7 cells with estradiol results inthe activation of c-Src kinase activity with resultant activation ofMAPK (Di Domenico, 1996; Migliaccio, 1996). More recently, Migliaccio(1998), has demonstrated in T47D breast cancer cells that progestinstimulated cell proliferation is dependent upon Src/Ras/MAPK signaling.The fact that these steroid receptors can utilize Ras for theirsignaling suggests that Ras may play a more pivotal role in thepromotion of growth in ER and PR positive breast tumors.

[0067] Ras itself may also play a more pivotal role in the developmentof breast cancer that was initially thought. Although activatingmutations in Ras are rare in breast cancer development, overexpressionof normal H-Ras has been observed in breast tumors (Shackney, 1998;Spandidos, 1987). This overexpression of Ras may provide an additionalmechanism of uncoupling normal signal transduction and promote atumorigenic state. Taken together these observations suggest thefeasibility of targeting an activated Ras pathway in the development ofnovel breast cancer therapeutics.

[0068] Previously we have demonstrated that reovirus is replicationcompetent in only those cells that contained an activated Ras signalingpathway, either through direct mutation of Ras itself or via upstreamelements that result in its activation (Strong, 1993; Strong, 1998).Additionally, we were able to demonstrate that reovirus could actagainst tumors containing a Ras activation in vivo (Coffey, 1998). Usingthe human glioblastoma cell line U87, we established a tumor xenograftSCID mouse model. U87 cells were selected as an appropriate model asthey overexpress the RTK PDGF that results in Ras activation anddemonstrate acute susceptibility to reovirus infection in vitro. SCIDmice were implanted with a U87 tumor xenograft and following palpabletumor establishment the tumors were treated with a single intratumorinjection of reovirus. This single treatment resulted in dramatic tumorregression.

[0069] In view of the drawbacks associated with the current means fortreating neoplastic growth, the need still exists for improved methodsfor the treatment of most types of cancers and, particularly, breastcancer. It would be useful to have a means available to determinereovirus susceptibility to predict the effectiveness of reovirustreatment.

SUMMARY OF THE INVENTION

[0070] The present invention is directed to methods of identifying thesusceptibility of cells to reovirus infection by measuring constitutiveras-MAP signaling. The invention also provides methods using reovirusfor the treatment of cellular proliferative disorders, and particularlycellular proliferative disorders wherein the proliferating cells exhibitconstitutive MAPK phosphorylation, in mammals. In particular, themethods provide for reovirus treatment of mammals to treat proliferativedisorders which include breast tumors, a subset of tumors in whichmutation of the ras gene is not believed to play a significant role.

[0071] This invention also pertains to methods for preventing reovirusrecognition in the treatment of cellular proliferative disorders, andparticularly cellular proliferative disorders which exhibit constitutiveMAPK phosphorylation, in mammals. The mammal may be selected from dogs,cats, sheep, goats, cattle, horses, pigs, mice, humans and non-humanprimates. The method comprises administering to the proliferating cellsan effective amount of one or more reoviruses under conditions whichresult in substantial lysis of the proliferating cells.

[0072] The reovirus may be a mammalian reovirus or an avian reovirus.The reovirus may be modified such that the outer capsid is removed, thevirion is packaged in a liposome or micelle or the proteins of the outercapsid have been mutated. The reovirus agent can be administered in asingle dose or in multiple doses. The proliferative disorder may be aneoplasm. Both solid and hematopoietic neoplasms can be targeted. Use ofpreceding, concurrent or subsequent immunosuppression may result in moreeffective reovirus treatment.

[0073] Accordingly, in one aspect the invention provides a method ofdetermining cellular susceptibility to reovirus infection by measuringconstitutive ras-MAP signaling, wherein the presence of said signalingindicates susceptibility to infection by reovirus. Constitutive ras-MAPsignaling results in activation of the MAP kinase whether there is amitogen or not, and activation of the MAP kinase leads tophosphorylation of MAP kinase. Therefore, the state of MAP kinasephosphorylation can be determined as a measure of constitutive ras-MAPsignaling. The state of MAP kinase phosphorylation can be determined byany method established in the art, and in particular by using anantibody specific for phosphorylated MAP kinase.

[0074] Accordingly, the present invention can be used to diagnoseproliferative disorders which can be treated with reovirus. Thus, abiological sample can be collected from a mammal suspected of having aproliferative disorder and cells in the sample are tested forconstitutive ras-MAP signaling according to the present invention. Theproliferative disorder may be any condition associated with abnormallyactive cell growth, such as neurofibromatosis. More preferably, theproliferative disorder is selected from the group consisting of lungcancer, prostate cancer, colorectal cancer, thyroid cancer, renalcancer, adrenal cancer, liver cancer, pancreatic cancer, breast cancerand central and peripheral nervous system cancer. A particularlypreferred proliferative disorder is breast cancer.

[0075] The present invention is applicable to any animal with aproliferative disorder. Preferably, the animal is a mammal. Morepreferably, the animal is selected from the group consisting of dogs,cats, sheep, goats, cattle, horses, pigs, mice, non-human primates, andhumans. Most preferably, the animal is human.

[0076] In a further aspect, the invention provides a method of treatinga proliferative disorder in a mammal, which disorder is characterized byproliferating cells which exhibit constitutive MAPK phosphorylation,comprising administering to the proliferating cells in said mammal aneffective amount of one or more reoviruses under conditions which resultin substantial lysis of the proliferating cells. Methods which furthercomprise a step selected from the group consisting of: administering tothe proliferating cells in said mammal an effective amount of an immunesuppressive agent; removing B-cells or T-cells from said mammal;removing anti-reovirus antibodies from said mammal; removing antibodiesfrom said mammal; administering anti-antireovirus antibodies to saidmammal; and suppressing the immune system of the mammal are alsoprovided, as are methods which further comprise administration of achemotherapeutic agent.

[0077] Also provided is a method of treating a neoplasm in a human,which neoplasm is characterized by proliferating cells which exhibitconstitutive MAPK phosphorylation, comprising administering to theneoplasm a reovirus in an amount sufficient to result in substantialoncolysis of the neoplastic cells. Preferably, the reovirus isadministered systemically or by injection into or near a solid neoplasm.Methods further comprising the step of previously, concurrently orsubsequently suppressing or otherwise compromising the immune system ofthe mammal are also included.

[0078] Also provided is a method of inhibiting metastasis of a neoplasmin a mammal, which neoplasm is characterized by proliferating cellswhich exhibit constitutive MAPK phosphorylation, comprisingadministering to the mammal a reovirus in an amount sufficient to resultin substantial lysis of the neoplastic cells. Methods further comprisingthe step of previously, concurrently or subsequently suppressing orotherwise compromising the immune system of the mammal are alsoincluded.

[0079] Also provided is a method of treating a suspected neoplasm in amammal, which neoplasm is characterized by proliferating cells whichexhibit constitutive MAPK phosphorylation, comprising surgical removalof substantially all of the neoplasm and administration of an effectiveamount of reovirus at or near to the surgical site resulting inoncolysis of any remaining neoplastic cells. The reovirus may also beadministered systemically. Methods further comprising the step ofpreviously, concurrently or subsequently suppressing or otherwisecompromising the immune system of the mammal are also included.

[0080] Also provided is a pharmaceutical composition comprising aneffective amount of a reovirus and a pharmaceutically acceptableexcipient. Also provided is a pharmaceutical composition comprising animmunosuppressant or immunoinhibitant, a reovirus and a pharmaceuticallyacceptable excipient. Kits comprising a reovirus and, optionally, animmunosuppressant or immunoinhibitant are also provided.

[0081] The methods and pharmaceutical compositions of the inventionprovide an effective means to treat neoplasia, without the side effectsassociated with other forms of cancer therapy. When used, inhibition orsuppression of the immune system increases the availability of reovirusto infect and lyse proliferating cells which exhibit constitutive MAPKphosphorylation because anti-reovirus antibodies are not formed. Becausereovirus is not known to be associated with disease, any safety concernsassociated with deliberate administration of a virus are minimized.

[0082] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0083]FIG. 1 shows the effect of v-src transformation on host cellsusceptibility to reovirus infection. Uninfectable NIH-3T3 parentalcells and v-src transformed NIH-3T3 cells were grown to 80% confluencyin a 24 well plate and were then exposed to reovirus at an estimated MOIof 80 PFU per cell. Cells and media were harvested at the indicatedtimes post infection and resultant lysate was used for plaque titrationassay. V-src transformed NIH-3T3 cells (closed circles), NIH-3T3parentals (open circles). (Mean ± standard deviation).

[0084]FIGS. 2A to 2 C illustrate in vitro reovirus replication in humanbreast tumor cell lines. (A) Reovirus protein synthesis in mock-infectedand reovirus-infected breast cancer cell lines. Cells were labeled with[³⁵S]-methionine from 46 to 48 hours after infection. Lysates wereprepared and subsequently immunoprecipitated with a polyclonalanti-reovirus type 3 serum and then analyzed by SDS-PAGE. Reovirusproteins are indicated on the right. (B) Reovirus infectivity correlateswith constitutive MAPK phosphorylation. The breast tissue cell lineHBL-100 and the breast tumor cell lines MDA-MB-468, MCF7, MDA-MB-435,T47D and SK-BR-3 were plated in a six well plate. Cells were eithergrown in the presence of 10% FCS or were serum starved (0.5% FCS) for a48 hour period. Monolayers were washed in PBS and cell lysates wereprepared and subjected to SDS-PAGE. Following blotting ontonitrocellulose paper, samples were probed with antibodies directedagainst phospho-MAPK Phospho-MAPK levels were all standardized by totalMAPK levels. (C) Total MAPK levels in the human breast tissue and breastcancer cell lines.

[0085]FIG. 3 illustrates reovirus-mediated in vivo oncolysis againsthuman breast tumor xenograft. SCID mice were implanted subcutaneouslyand unilaterally with MDA-MB-468 human breast tumor xenografts.Following the establishment of a palpable tumor mass, a singleintratumoral injection of either live reovirus (open circles) orUV-inactivated virus (closed circles) was administered and tumor growthwas followed for a period of four weeks. (Mean ± SEM).

DETAILED DESCRIPTION OF THE INVENTION

[0086] Previously we have shown that the human reovirus could be used asan effective oncolytic agent against human glioblastoma xenografts in aSCID mouse model (WO 99/08692). Here we have found that cellularsusceptibility to reovirus infection may be determined by measuringconstitutive ras-MAP signaling of the cell, since the presence of suchsignaling indicates susceptibility to infection by reovirus. We alsopresent evidence that reovirus is useful as an oncolytic agent againstmammary tumors. Although ras mutations are infrequent in the etiology ofbreast cancer, aberrant Ras/MAPK signaling via upstream signal elements,such as receptor tyrosine kinases and non-receptor tyrosine kinases(such as c-Src), is common.

[0087] For the same reasons that the treatment of breast cancers withmonoclonal antibodies may be impaired (see discussion above), reovirusmay be an attractive cancer therapy. Firstly, reovirus itself is acytocidal agent and does not rely upon immune effector cells to causetumor regression. Indeed, the natural mechanism of killing infectedcells is via direct lysis due to viral replication (Tyler and Fields,1996). Secondly, it targets those breast cancers in which there isactivation of Ras. This activation is not restricted to activatingmutations of Ras (admittedly a rare subset of breast tumors) but alsoincludes activation of Ras caused by elements upstream of Ras itself.These elements include not only receptor tyrosine kinases such as EGFRand HER2, but also include non-receptor tyrosine kinases such as the Srcfamily members. Taken together, this type of therapy could be used withgreat efficacy against a tumor type as heterogeneous as breast cancerand would not be as restricted a strategy as one that targets only onreceptor. Finally, the inability of antibodies to penetrate into solidtumor masses suggests that reovirus, if delivered intratumorally, shouldreplicate unheeded. Thus, it is useful to know whether the cells of aproliferative disorder are susceptible to infection by reovirus in orderto predict the efficacy of such treatment.

[0088] To assess whether Src family kinases could mediate reovirusinfection, uninfectable NIH-3T3 cells were transformed with v-src andwere then challenged with reovirus. We determined that v-src signalingconfers infectability to reovirus infection.

[0089] We next examined five breast cancer cell lines; MDA-MB-468, MCF7,MDA-MB-435, T47D, and SK-BR-3 as well as a cell line derived from normalbreast tissue, HBL-100 for in vitro reovirus replication. All five ofthe tumor derived cell lines were infectable by reovirus while theHBL-100 was unable to effectively replicate the virus.

[0090] To determine if the Ras pathway was indeed activated in thesecell lines, the level of MAPK phosphorylation was assessed in thepresence and absence of serum. Those cell lines that were infectableexhibited constitutive MAPK phosphorylation even in the absence ofmitogen, while the cell line which was not susceptible to reovirusinfection displayed MAPK phosphorylation only in the presence of serum.Therefore, constitutive MAPK phosphorylation is an indication ofsusceptibility to reovirus infection.

[0091] To determine if reovirus could be used as an oncolytic agent invivo against breast tumors, SCID mice were implanted with MDA-MB-468human tumour xenographs. Following the establishment of palpabletumours, the mice were treated with a single injection of reovirus andtumour size was monitored for a four-week period. The single injectionresulted in dramatic regression of the tumour size. Finally, the abilityof reovirus to act against primary breast cancer tumours was determined,and the results indicate that reovirus was capable of replicating inbiopsy samples collected from various patients. Therefore, many breasttumors are susceptible to reovirus infection.

[0092] The name reovirus (Respiratory and enteric orphan virus) is adescriptive acronym suggesting that these viruses, although notassociated with any known disease state in humans, can be isolated fromboth the respiratory and enteric tracts (Sabin, A. B. (1959), Science130:966). The term “reovirus” refers to all viruses classified in thereovirus genus.

[0093] Reoviruses are viruses with a double-stranded, segmented RNAgenome.

[0094] The virions measure 60-80 nm in diameter and possess twoconcentric capsid shells, each of which is icosahedral. The genomeconsists of double-stranded RNA in 10-12 discrete segments with a totalgenome size of 16-27 kbp. The individual RNA segments vary in size.Three distinct but related types of reovirus have been recovered frommany species. All three types share a common complement-fixing antigen.

[0095] The human reovirus consists of three serotypes: type 1 (strainLang or T1L), type 2 (strain Jones, T2J) and type 3 (strain Dearing orstrain Abney, T3D). The three serotypes are easily identifiable on thebasis of neutralization and hemagglutinin-inhibition assays (Sabin, A.B. (1959), Science 130:966; Fields, B. N. et al. (1996), FundamentalVirology, 3rd Edition, Lippincott-Raven; Rosen, L. (1960) Am. J.Hyg.71:242; Stanley, N. F. (1967) Br. Med. Bull. 23:150).

[0096] Although reovirus is not known to be associated with anyparticular disease, many people have been exposed to reovirus by thetime they reach adulthood (i.e., fewer than 25% in children <5 yearsold, to greater than 50% in those 20-30 years old (Jackson G. G. andMuldoon R. L. (1973) J. Infect. Dis. 128:811; Stanley N. F. (1974) In:Comparative Diagnosis of Viral Diseases, edited by E. Kurstak and K.Kurstak, 385-421, Academic Press, New York).

[0097] For mammalian reoviruses, the cell surface recognition signal issialic acid (Armstrong, G. D. et al. (1984), Virology 138:37; Gentsch,J. R. K. and Pacitti, A. F. (1985), J. Virol. 56:356; Paul R. W. et al.(1989) Virology 172:382-385) Due to the ubiquitous nature of sialicacid, reovirus binds efficiently to a multitude of cell lines and assuch can potentially target many different tissues; however, there aresignificant differences in susceptibility to reovirus infection betweencell lines.

[0098] As described herein, Applicant have discovered that cells whichexhibit constitutive MAPK phosphorylation are susceptible to reovirusinfection. “Resistance” of cells to reovirus infection indicates thatinfection of the cells with the virus did not result in significantviral production or yield. Cells that are “susceptible” are those thatdemonstrate induction of cytopathic effects, viral protein synthesis,and/or virus production. Resistance to reovirus infection was found tobe at the level of gene translation, rather than at early transcription:while viral transcripts were produced, virus proteins were notexpressed.

[0099] The implantation of human tumor cells into SCID mice isrecognized as a well known model system for testing the effectiveness ofvarious anti-tumor agents in humans. It has previously been shown thatpharmaceuticals effective against human tumors implanted into SCID miceare predictive of their effectiveness against the same tumors in humans.

[0100] Based upon these discoveries, Applicant have developed methodsfor determining susceptibility to reovirus infection by measuringconstitutive ras-MAP signaling and methods for treating proliferativedisorders in mammals wherein the proliferating cells exhibitconstitutive MAPK phosphorylation. Representative mammals include dogs,cats, sheep, goats, cattle, horses, pigs, mice, non-human primates, andhumans. In a preferred embodiment, the mammal is a human.

[0101] In the diagnostic methods of the invention, the level of MAPKphosphorylation of proliferating cells is determined in the presence orabsence of mitogen. The presence of such constitutive ras-MAP signalingin the cells is indicative of susceptibility to reovirus infection.

[0102] In the treatment methods of the invention, reovirus isadministered to proliferating cells in the individual mammal exhibitingconstitutive MAPK phosphorylation. In one embodiment of this invention acourse of reovirus therapy is administered one or more times.

[0103] In the methods of the invention, reovirus is administered toproliferating cells in the individual mammal exhibiting constitutiveMAPK phosphorylation. Representative types of human reovirus that can beused include type 1 (e.g., strain Lang or T1L); type 2 (e.g., strainJones or T2J); and type 3 (e.g., strain Dearing or strain Abney, T3D orT3A); other strains of reovirus can also be used. In a preferredembodiment, the reovirus is human reovirus serotype 3, more preferablythe reovirus is human reovirus serotype 3, strain Dearing.Alternatively, the reovirus can be a non-human mammalian reovirus (e.g.,non-human primate reovirus, such as baboon reovirus; equine; or caninereovirus), or a non-mammalian reovirus (e.g., avian reovirus). Acombination of different serotypes and/or different strains of reovirus,such as reovirus from different species of animal, can be used.

[0104] The reovirus may be naturally occurring or modified. The reovirusis “naturally-occurring ”: when it can be isolated from a source innature and has not been intentionally modified by humans in thelaboratory. For example, the reovirus can be from a “field source”: forexample, from a patient.

[0105] The reovirus may be modified but still capable of lyticallyinfecting a mammalian cell exhibiting constitutive MAPK phosphorylation.The reovirus may be chemically or biochemically pretreated (e.g., bytreatment with a protease, such as chymotrypsin or trypsin) prior toadministration to the proliferating cells. Pretreatment with a proteaseremoves the outer coat or capsid of the virus and may increase theinfectivity of the virus. The reovirus may be coated in a liposome ormicelle (Chandron and Nibert, “Protease cleavage of reovirus capsidprotein mul and mulC is blocked by alkyl sulfate detergents, yielding anew type of infectious subvirion particle” J. of Virology 72(1):467-75(1998)) to reduce or prevent an immune response from a mammal which hasdeveloped immunity to the reovirus. For example, the virion may betreated with chymotrypsin in the presence of micelle formingconcentrations of alkyl sulfate detergents to generate a new infectioussubviral particle (ISVP). An ISVP may be used either alone or incombination with whole virus to provide an agent that is either poorlyrecognized or has not been previously prevented by the patient's immunesystem.

[0106] The reovirus may be a recombinant reovirus from two or more typesof reoviruses with differing pathogenic phenotypes such that it containsdifferent antigenic determinants thereby reducing or preventing animmune response by a mammal previously exposed to a reovirus subtype.Such recombinant virions, also known as reassortants, can be generatedby co-infection of mammalian cells with different subtypes of reoviruswith the resulting resorting and incorporation of different subtype coatproteins into the resulting virion capsids.

[0107] The reovirus may be modified by incorporation of mutated coatproteins, such as for example o1, into the virion outer capsid. Theproteins may be mutated by replacement, insertion or deletion.Replacement includes the insertion of different amino acids in place ofthe native amino acids. Insertions include the insertion of additionalamino acid residues into the protein at one or more locations. Deletionsinclude deletions of one or more amino acid residues in the protein.Such mutations may be generated by methods known in the art. Forexample, oligonucleotide site directed mutagenesis of the gene encodingfor one of the coat proteins could result in the generation of thedesired mutant coat protein. Expression of the mutated protein inreovirus infected mammalian cells in vitro such as COS1 cells willresult in the incorporation of the mutated protein into the reovirusvirion particle (Turner and Duncan, “Site directed mutagenesis of theC-terminal portion of reovirus protein sigma1: evidence for aconformation-dependent receptor binding domain” Virology 186(1):219-27(1992); Duncan et al., “Conformational and functional analysis of theC-terminal globular head of the reovirus cell attachment protein”Virology 182(2):810-9 (1991); Mah et al., “The N-terminal quarter ofreovirus cell attachment protein sigma 1 possesses intrinsicvirion-anchoring function”. Virology 179(1):95-103 (1990))

[0108] The reovirus is preferably a reovirus modified to reduce oreliminate an immune reaction to the reovirus. Such modified reovirus aretermed “immunoprotected reovirus” Such modifications could includepackaging of the reovirus in a liposome, a micelle or other vehicle tomask the reovirus from the mammals immune system. Alternatively, theouter capsid of the reovirus virion particle may be removed since theproteins present in the outer capsid are the major determinant of thehost humoral and cellular responses.

[0109] A “proliferative disorder” is any cellular disorder in which thecells proliferate more rapidly than normal tissue growth. Thus a“proliferating cell” is a cell that is proliferating more rapidly thannormal cells. The proliferative disorder, includes but is not limited toneoplasms. A neoplasm is an abnormal tissue growth, generally forming adistinct mass, that grows by cellular proliferation more rapidly thannormal tissue growth. Neoplasms show partial or total lack of structuralorganization and functional coordination with normal tissue. These canbe broadly classified into three major types. Malignant neoplasmsarising from epithelial structures are called carcinomas, malignantneoplasms that originate from connective tissues such as muscle,cartilage, fat or bone are called sarcomas and malignant tumorsaffecting hematopoietic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias and lymphomas. A tumor is the neoplastic growth of thedisease cancer. As used herein, a “neoplasm”, also referred to as a“tumor”, is intended to encompass hematopoietic neoplasms as well assolid neoplasms. Other proliferative disorders include, but are notlimited to neurofibromatosis.

[0110] At least some of the cells of the proliferative disorder whichare treated using the methods of the present invention exhibitconstitutive MAPK phosphorylation. Similarly, at least some of the cellswhich are determined to be susceptible to reovirus infection using themethods of the present invention exhibit constitutive MAPKphosphorylation.

[0111] “B-cells” refers to B-lymphocytes. There are two majorsubpopulations of B lymphocytes, B-1 and B-2 cells. B-1 cells areself-renewing and frequently secrete high levels of antibody which bindsto a range of antigens (polyspecificity) with a relatively low affinity.The majority of B cells, B-2 cells, are directly generated fromprecursors in the bone marrow and secrete highly specific antibody.

[0112] “T-cells” refers to T-lymphocytes. T-cells differentiate withinthe thymus gland and are specialized to operate against cells bearingintracellular organisms. T-cells only recognize antigen when it is onthe surface of a body cell.

[0113] “Anti-reovirus antibody” refers to an antibody which binds toreovirus. IgG antibodies” refers to immunoglobulin G antibodies. IgG,the most abundant type of antibody, carries the major burden ofneutralizing bacterial toxins and binding to microorganisms to enhancetheir phagocytosis. “Humanized antibodies” refers to antibody moleculesin which the amino acid sequence in the non-antigen binding regions hasbeen altered so that the antibody more closely resembles a humanantibody, and still retains its original binding ability.

[0114] “Administration to a proliferating cell or neoplasm” indicatesthat the reovirus is administered in a manner so that it contacts theproliferating cells or cells of the neoplasm (also referred to herein as“neoplastic cells”). The route by which the reovirus is administered, aswell as the formulation, carrier or vehicle, will depend on the locationas well as the type of the neoplasm. A wide variety of administrationroutes can be employed. For example, for a solid neoplasm that isaccessible, the reovirus can be administered by injection directly tothe neoplasm. For a hematopoietic neoplasm, for example, the reoviruscan be administered intravenously or intravascularly. For neoplasms thatare not easily accessible within the body, such as metastases or braintumors, the reovirus is administered in a manner such that it can betransported systemically through the body of the mammal and therebyreach the neoplasm (e.g., intrathecally, intravenously orintramuscularly). Alternatively, the reovirus can be administereddirectly to a single solid neoplasm, where it then is carriedsystemically through the body to metastases. The reovirus can also beadministered subcutaneously, intraperitoneally, topically (e.g., formelanoma), orally (e.g., for oral or esophageal neoplasm), rectally(e.g., for colorectal neoplasm), vaginally (e.g., for cervical orvaginal neoplasm), nasally or by inhalation spray (e.g., for lungneoplasm).

[0115] Reovirus can be administered systemically to mammals which areimmune compromised or which have not developed immunity to the reovirusepitopes. In such cases, reovirus administered systemically, i.e. byintraveneous injection, will contact the proliferating cells resultingin lysis of the cells. Where the mammals to be treated have highertiters of anti-reovirus antibodies, more reovirus must be administeredto be effective.

[0116] Immunocompetent mammals previously exposed to a reovirus subtypemay have developed humoral and/or cellular immunity to that reovirussubtype. Nevertheless, it has been found that direct injection of thereovirus into a solid tumor in immunocompetent mammals will result inthe lysis of the neoplastic cells. On the other hand, when the reovirusis administered systemically to immunocompetent mammals, the mammals mayproduce an immune response to the reovirus. Such an immune response maybe avoided if the reovirus is of a subtype to which the mammal has notdeveloped immunity, or the reovirus has been modified as previouslydescribed herein such that it is immunoprotected, for example, byprotease digestion of the outer capsid or packaging in a micelle.

[0117] Alternatively, it is contemplated that the immunocompetency ofthe mammal against the reovirus may be suppressed either by the prior orco-administration of pharmaceuticals known in the art to suppress theimmune system in general (Cuff et al., “Enteric reovirus infection as aprobe to study immunotoxicity of the gastrointestinal tract”Toxicological Sciences 42(2):99-108 (1998)) or alternatively theadministration of such immunoinhibitors as anti-antireovirus antibodies.The humoral immunity of the mammal against reovirus may also betemporarily reduced or suppressed by plasmaphoresis of the mammals bloodto remove the anti-reovirus antibodies. The humoral immunity of themammal against reovirus may additionally be temporarily reduced orsuppressed by the intraveneous administration of non-specificimmunoglobulin to the mammal.

[0118] It is contemplated that the reovirus may be administered toimmunocompetent mammals immunized against the reovirus in conjunctionwith the administration of immunosuppressants and/or immunoinhibitors.Such immunosuppressants and immunoinhibitors are known to those of skillin the art and include such agents as cyclosporin, rapamycin,tacrolimus, mycophenolic acid, azathioprine and their analogs, and thelike. Other agents are known to have immunosuppressant properties aswell (see, e.g., Goodman and Gilman, 7^(th) Edition, page 1242, thedisclosure of which is incorporated herein by reference). Suchimmunoinhibitors also include “anti-antireovirus antibodies, ” which areantibodies directed against anti-reovirus antibodies. Such antibodiescan be made by methods known in the art. See for example “Antibodies: Alaboratory manual” E. Harlow and D. Lane, Cold Spring Harbor Laboratory(1988). Such anti-antireovirus antibodies may be administered prior to,at the same time or shortly after the administration of the reovirus.Preferably an effective amount of the anti-antireovirus antibodies areadministered in sufficient time to reduce or eliminate an immuneresponse by the mammal to the administered reovirus. The terms“immunosuppressant” or “immune suppressive agent” include conventionalimmunosuppressants, immunoinhibitors, antibodies, and conditions such asradiation therapy or HIV infection which result in compromise of theimmune system.

[0119] The term “substantial lysis” means at least 10% of theproliferating cells are lysed, more preferably of at least 50% and mostpreferably of at least 75% of the cells are lysed. The percentage oflysis can be determined for tumor cells by measuring the reduction inthe size of the tumor in the mammal or the lysis of the tumor cells invitro.

[0120] A “mammal suspected of having a proliferative disorder” meansthat the mammal may have a proliferative disorder or tumor or has beendiagnosed with a proliferative disorder or tumor or has been previouslydiagnosed with a proliferative disorder or tumor, the tumor orsubstantially all of the tumor has been surgically removed and themammal is suspected of harboring some residual tumor cells.

[0121] This invention also includes pharmaceutical compositions whichcontain, as the active ingredient, one or more immunosuppressants orimmunoinhibitors and one or more of the reoviruses associated with“pharmaceutically acceptable carriers or excipients”. In making thecompositions of this invention, the active ingredients/immunosuppressantor immunoinhibitor and reovirus are usually mixed with an excipient,diluted by an excipient or enclosed within such a carrier which can bein the form of a capsule, sachet, paper or other container. When thepharmaceutically acceptable excipient serves as a diluent, it can be asolid, semi-solid, or liquid material, which acts as a vehicle, carrieror medium for the active ingredient. Thus, the compositions can be inthe form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

[0122] Some examples of suitable excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, andmethyl cellulose. The formulations can additionally include: lubricatingagents such as talc, magnesium stearate, and mineral oil; wettingagents; emulsifying and suspending agents; preserving agents such asmethyl- and propylhydroxy-benzoates; sweetening agents; and flavoringagents. The compositions of the invention can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the patient by employing procedures known in theart.

[0123] For preparing solid compositions such as tablets, the principalactive ingredients/immunosuppressant or immunoinhibitor and reovirus aremixed with a pharmaceutical excipient to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention. When referring to these preformulation compositionsas homogeneous, it is meant that the active ingredient is dispersedevenly throughout the composition so that the composition may be readilysubdivided into equally effective unit dosage forms such as tablets,pills and capsules.

[0124] The tablets or pills of the present invention may be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by anenteric layer which serves to resist disintegration in the stomach andpermit the inner component to pass intact into the duodenum or to bedelayed in release. A variety of materials can be used for such entericlayers or coatings, such materials including a number of polymeric acidsand mixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

[0125] The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

[0126] Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedherein. Preferably the compositions are administered by the oral ornasal respiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

[0127] Another preferred formulation employed in the methods of thepresent invention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the reovirus of the present invention in controlled amounts.The construction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, for example, U.S.Pat. No. 5,023,252, herein incorporated by reference. Such patches maybe constructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

[0128] Other suitable formulations for use in the present invention canbe found in Remington's Pharmaceutical Sciences, the disclosure of whichis incorporated herein by reference.

[0129] The immunosuppressant or immunoinhibitor and reovirus or thepharmaceutical composition comprising the immunosuppressant orimmunoinhibitor and reovirus may be packaged into convenient kitsproviding the necessary materials packaged into suitable containers. Itis contemplated the kits may also include chemotherapeutic agent.

[0130] The immunosuppressant or immunoinhibitor is administered in anappropriate amount and using an appropriate schedule of administrationsufficient to result in immunosuppression or immunoinhibition of themammal's immune system. Such amounts and schedules are well known tothose of skill in the art.

[0131] The reovirus is administered in an amount that is sufficient totreat the proliferative disorder (e.g., an “effective amount”). Aproliferative disorder is “treated” when administration of reovirus tothe proliferating cells effects lysis of the proliferating cells. Thismay result in a reduction in size of the neoplasm, or in a completeelimination of the neoplasm. The reduction in size of the neoplasm, orelimination of the neoplasm, is generally caused by lysis of neoplasticcells (“oncolysis”) by the reovirus. Preferably the effective amount isthat amount able to inhibit tumor cell growth. Preferably the effectiveamount is from about 1.0 pfu/kg body weight to about 10¹⁵ pfu/kg bodyweight, more preferably from about 10² pfu/kg body weight to about 10¹³pfu/kg body weight. For example, for treatment of a human, approximately10² to 10¹⁷ plaque forming units (PFU) of reovirus can be used,depending on the type, size and number of tumors present. The effectiveamount will be determined on an individual basis and may be based, atleast in part, on consideration of the type of reovirus; the chosenroute of administration; the individual's size, age, gender; theseverity of the patient's symptoms; the size and other characteristicsof the neoplasm; and the like. The course of therapy may last fromseveral days to several months or until diminution of the disease isachieved.

[0132] The immunosuppressant or immunoinhibitor and reovirus can beadministered in a single dose, or multiple doses (i.e., more than onedose). The multiple doses can be administered concurrently, orconsecutively (e.g., over a period of days or weeks). The reovirus canalso be administered to more than one neoplasm in the same individual.

[0133] The compositions are preferably formulated in a unit dosage form,each dosage containing an appropriate amount of immunosuppressant orimmunoinhibitor and from about 10² pfus to about 10¹³ pfus of thereovirus. The term “unit dosage forms” refers to physically discreteunits suitable as unitary dosages for human subjects and other mammals,each unit containing a predetermined quantity of reovirus calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical excipient.

[0134] As mentioned above, it has been found that the reovirus iseffective for the treatment of solid neoplasms in immunocompetentmammals. Administration of unmodified reovirus directly to the neoplasmresults in oncolysis of the neoplastic cells and reduction in the sizeof the tumor in immunocompetent animals. When animals are renderedimmunosuppressed or immunodeficient in some way, systemic administrationof reovirus will be more effective in producing oncolysis.

[0135] It is contemplated that the reovirus may be administered inconjunction with surgery or removal of the neoplasm. Therefore, providedherewith are methods for the treatment of a solid neoplasm comprisingsurgical removal of the neoplasm and administration of a reovirus at ornear to the site of the neoplasm.

[0136] It is contemplated that the reovirus may be administered inconjunction with or in addition to radiation therapy which renders themammal immunosuppressed.

[0137] It is further contemplated that the reovirus of the presentinvention may be administered in conjunction with or in addition toknown anticancer compounds or chemotherapeutic agents. Chemotherapeuticagents are compounds which may inhibit the growth of tumors. Suchagents, include, but are not limited to, 5-fluorouracil, mitomycin C,methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone,anthracyclins (Epirubicin and Doxurubicin), antibodies to receptors,such as herceptin, etopside, pregnasome, platinum compounds such ascarboplatin and cisplatin, taxanes such as taxol and taxotere, hormonetherapies such as tamoxifen and anti-estrogens, interferons, aromataseinhibitors, progestational agents and LHRH analogs.

[0138] The reovirus and immunosuppressants of the present invention havebeen found to reduce the growth of tumors that are metastatic. In anembodiment of the invention, a method is provided for reducing thegrowth of metastatic tumors in a mammal comprising administering aneffective amount of a reovirus to the immunosuppressed mammal.

[0139] Utility

[0140] The diagnostic methods of the present invention may be used toidentify the susceptibility of cells to reovirus infection by measuringconstitutive ras-MAP signaling. This will be useful to determine in whatcases reovirus treatment of cellular proliferative disorders is likelyto be effective.

[0141] The reoviruses and immunosuppressants of the present inventionmay be used for a variety of purposes. They may be used in methods fortreating proliferative disorders which exhibit constitutive MAPKphosphorylation in a mammal. They may be used to reduce or eliminateneoplasms. They may be used in methods for treating metastases. They maybe used in conjunction with known treatments for cancer includingsurgery, chemotherapy and radiation.

[0142] In order to further illustrate the present invention andadvantages thereof, the following specific examples are given but arenot meant to limit the scope of the claims in any way.

EXAMPLES

[0143] In the examples below, all temperatures are in degrees Celsius(unless otherwise indicated) and all percentages are weight percentages(also unless otherwise indicated).

[0144] In the examples below, the following abbreviations have thefollowing meanings. If an abbreviation is not defined, it has itsgenerally accepted meaning: μM = micromolar mM = millimolar M = molar ml= milliliter μl = microliter mg = milligram μg = microgram PAGE =polyacrylamide gel electrophoresis rpm = revolutions per minute FBS =fetal bovine serum DTT = dithiothrietol SDS = sodium dodecyl sulfate PBS= phosphate buffered saline DMEM = Dulbecco's modified Eagle's mediumα-MEM = α-modified Eagle's medium β-ME = β-mercaptoethanol MOI =multiplicity of infection PFU = plaque forming units MAPK = MAP kinasephosph-MAPK = phosphorylated-MAP kinase HRP = horseradish-peroxidase PKR= double-stranded RNA activated protein kinase RT-PCR = reversetranscriptase-polymerase chain reaction GAPDH =glyceraldehyde-3-phosphate dehydrogenase EGFR = epidermal growth factorreceptors MEK kinase = mitogen-activated extracellular signal-regulatedkinase DMSO = dimethylsulfoxide SCID = severe combined immunodeficiency

[0145] General Methods

[0146] Cells and Virus

[0147] Parental NIH-3T3 cells along with NIH-3T3 cells transformed withv-Src were a generous gift of Dr. Jove (University of Florida). Breasttumor cell lines MDA-MB-468, MCF7, MDA-MB-435, T-47D, SK-BR-3 and thecontrol HBL-100 cells, were a generous gift of Dr. Karl Riabowol(University of Calgary). All cell lines were grown in Dulbecco'smodified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS).

[0148] The Dearing strain of reovirus serotype 3 used in these studieswas propagated in suspension cultures of L cells and purified accordingto Smith et al (1969) with the exception that β-mercaptoethanol (β-ME)was omitted from the extraction buffer.

[0149] Infection of Cells and Quantitation of Virus

[0150] Confluent monolayers of cells were grown in 24-well plates andinfected with reovirus at an estimated multiplicity of infection of 80PFU/cell. After 1 hr incubation at 37° C., the monolayers were washedwith warm DMEM-10% FBS, and then incubated in the same medium. Atvarious times postinfection, a mixture of NP-40 and sodium deoxycholatewas added directly to the medium on the infected monolayers to finalconcentrations of 1% and 0.5% respectively. The lysates were thenharvested and virus yields were determined by plaque titration on L-929cells.

[0151] Radiolabelling of reovirus-infected cells and preparation oflysates

[0152] Subconfluent monolayers (80% confluent) of cells were infectedwith reovirus (MOI˜10 PFU/cell). At 46 hours after infection, the mediawas replaced with methionine-free DMEM containing 10% FBS and 0.1 mCi/ml[³⁵S]-methionine. After further incubation for 2 hours at 37° C., thecells were washed in phosphate-buffered saline (PBS) and lysed in thesame buffer containing 1% Triton X-100, and 0.5% sodium deoxycholate.Lysates were then boiled and stored at −70° C. until use.

[0153] Immunoprecipitation and SDS-PAGE analysis

[0154] Immunoprecipitation of ³⁵S-labeled reovirus-infected cell lysateswith anti-reovirus serotype 3 serum was carried out as previouslydescribed (Lee, P. W. K. et al. (1981) Virology, 108:134-146).

[0155] Detection of Phospho-MAPK and Total MAPK

[0156] The ‘PhosphoPlus’ p44/42 MAP kinase (Thr202/Tyr204) antibody kit(New England Biolabs) was used for the detection of MAP kinase in celllysates according to the manufacturer's instructions. Briefly,subconfluent monolayer cultures were lysed with the recommendedSDS-containing sample buffer, and subjected to SDS-PAGE, followed byelectroblotting onto nitrocellulose paper. The membrane was then probedwith the primary antibody (anti-total MAPK or anti-phospho-MAPK),followed by the horseradish peroxidase (HRP)-conjugated secondaryantibody as described in the manufacturer's manual.

[0157] Severe Combined Immune Deficient (SCID) Mice

[0158] Five to eight week old male SCID mice were purchased from CharlesRiver Canada and were treated according to a protocol approved by theUniversity of Calgary Animal Care Committee.

[0159] Implantation of Allografts and Xenografts

[0160] Actively growing MDA-MB-468 human breast cancer cells wereharvested, washed, and resuspended in sterile PBS at a density of 2×10⁷cells/ml. 2.0 ×10⁶ cells in 100 μl were injected subcutaneously at asite overlying the hind flank. Implanted tumors were allowed to grow for2-3 weeks until palpable tumors of 0.5 ×0.5 cm were obtained.

[0161] Intratumoral Injection of Reovirus

[0162] Once the established tumors obtained a treatable size, a singleintratumoral injection of 1.0 ×10⁷ PFUs of either live or UV-inactivatedreovirus (serotype 3, strain Dearing) in 20 μl sterile PBS. Tumor sizewas measured twice weekly for a period of two to four weeks. Animalswere sacrificed when they displayed severe morbidity due to excessivetumor burden or any observable level of distress.

[0163] Histoimmunological Analysis of Reovirus Infection

[0164] Immunofluorescent analysis was carried out using formalin-fixed,paraffin embedded tumor sections mounted on cover slips. After removalof paraffin by xylene, the sections were rehydrated, and exposed to theprimary antibody (rabbit polyclonal anti-reovirus type 3 serum diluted1/100 in PBS) for 2 h at room temperature. Following three washes withPBS, the sections were exposed to the secondary antibody [goatanti-rabbit IgG (whole molecule)-fluorescein isothiocyanate conjugate(FITC) or depending on the experiment, the same concentration of Cy3,diluted 1/100 in PBS containing 10% goat serum and 0.005 % Evan's Bluecounterstain] for 1 h at room temperature. As an additionalcounterstain, the nuclear stain DAPI, was also used. Finally, the fixedand treated sections were washed three more times with PBS and then oncewith double- distilled water. Slides were then dried and mounted onslides in 90% glycerol containing 0.1% phenylenediamine, and viewed witha Zeiss Axiophot microscope on which a Carl Zeiss camera was mounted(the magnification for all pictures was 200X).

[0165] Reovirus Infection of Primary Breast Tumor Samples

[0166] Biopsy breast tumor samples were sterilized by immersion in 95%ethanol followed by several washes of sterile PBS. The sample was thensliced into small sections and placed in a 24 well plate containing DMEMwith 10% FCS. Reovirus was added (1×10⁸PFUs). At various times postinfection, samples were washed in sterile PBS and then fixed informalin. Samples were then embedded in paraffin and sectioned for usein immunohistochemical analysis using antibodies directed against totalreovirus proteins.

[0167] Example 1. Transformation by the v-src Non-receptor TyrosineKinase Family Member Confers Susceptibility to Reovirus Infection

[0168] Although we have previously demonstrated that transfection ofuninfectable NR6 and NIH-3T3 cells with receptor tyrosine kinases wassufficient to allow reovirus replication (Strong, 1993), whethertransfection of non-receptor tyrosine kinases could result in reovirussusceptibility was unknown. To determine whether activation of Srcfamily kinases, which frequently contribute to the uncontrolledproliferation of many breast cancers, results in sufficient Ras activityto enable reovirus replication, uninfectable NIH-3T3 cells weretransformed with v-src and susceptibility to reovirus was assessed.Confluent monolayers of v-src transformed NIH-3T3 or their parentalcells were exposed to reovirus at a multiplicity of infection (MOI) of50 plaque forming units (PFUs) of reovirus. Cells and media wereharvested at various times after infection and resultant samples wereused for plaque titration analysis to determine reovirus replication.Our results show dramatic cytopathic effect in the v-src transformedcells by 48 hours postinfection (data not shown) as well as an enhancedreovirus output as measured by plaque titration assay. These results(FIG. 1) show that even non-receptor tyrosine kinases could potentiallymediate reovirus infection and thus may be able to target an additionalsubset of breast tumors.

[0169] Example 2. Reovirus Can Infect a Panel of Human Breast CancerDerived Cell Lines in Which Constitutive Ras/MAPK signaling Can BeAssessed

[0170] To assess the feasibility of using reovirus against breastderived tumors a panel of breast cancer cells including: MDA-MB-468,MCF7, MDA-MB-435, T-47D, and SK-BR-3 was selected and susceptibility toreovirus infection in vitro was determined. As a control, HBL-100 cells,which are derived from normal breast tissue were used. These six celllines were grown to 80% confluency and were then challenged withreovirus at an MOI of 10. Cells were labeled with [³⁵S] methionine for atwo-hour period at 48 hours postinfection. Cells were then washed inphosphate-buffered saline and were lysed. The prepared lysates were thenused for immunoprecipitation using antibodies directed against totalreovirus protein. Immunoprecipitated proteins were analyzed by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Theresults (FIG. 2A) clearly demonstrate effective reovirus replicationwithin the cell lines derived from breast tumor, however reovirusreplication was restricted in the HBL-100 cell line. This suggests thatthese breast tumor cells are infectable as a result of an activation ofRas, either through a direct mutation or via an upstream signalingelement. It is significant in that it suggests that a high percentage ofbreast cancers could potentially be treatable by therapeutics thattarget Ras, although this specific activation is rare in this cancertype.

[0171] As MAPK phosphorylation is the inevitable result of signalingthrough Ras, the phosphorylated state of the protein should only beobserved in a cell line with normal Ras signaling if a mitogenicstimulus is present. In a cell line with normal Ras signaling, theremoval of these mitogenic stimuli should result in the cessation ofsignal through this pathway with a resultant dephosphorylation of MAPK.In a cell with aberrant Ras activity, either directly through mutationalactivation of Ras or alternatively via an upstream element, thephosphorylated form of MAPK should persist in either the presence or theabsence of serum. To establish that the observed infectability in thebreast tumor cell lines is a result of the activation of the Ras/MAPKsignaling pathway, Western blot analysis of these cell lines wasconducted using antibodies directed against phospho-MAPK. MDA-MB-468,MCF7, MDA-MB-435, T-47D, SK-BR-3 and HBL-100 cells were plated in a sixwell plate. Cells were then grown in 10% fetal calf serum (FCS) or wereserum starved (0.5% FCS) for 48 hours. Cells were then washed inphosphate buffered saline (PBS) and were harvested in protein samplebuffer. Cell lysates were subjected to SDS-PAGE and were subsequentlyblotted onto nitrocellulose paper and probed with anti-phospho-MAPKantibodies as recommended by the manufacturer. The results (FIG. 2B)clearly demonstrate that the only uninfectable cell line, HBL-100,possesses phospho-MAPK in the presence of serum and not in its absence,indicating that Ras signalling in this pathway is not aberrant. Theremaining infectable cell lines have phospho-MAPK independent of thepresence of mitogenic signals, as would be expected if they hadconstitutive activation of this pathway. Protein concentrations werestandardized using antibodies directed against total MAPK (FIG. 2C).

[0172] Example 3. Reovirus Can Act as an Anti-tumor Agent Against BreastTumors In Vivo.

[0173] The human breast cancer cell line, MDA-MB-468 was introduced astumor xenografts subcutaneously into a site overlying the hind flank ofSCID mice. Following palpable tumor establishment, the MDA-MB-468 tumorwas administered a single intratumoral injection of 1.0×10⁷ plaqueforming units (PFUs) of reovirus serotype 3 (strain Dearing) in PBS.Control animals were given an intratumoral injection of UV-inactivatedreovirus. Tumor growth was followed for a period of four weeks. As indemonstrated (FIG. 3), the reovirus treatment results in a dramaticregression in tumor size. As before, hematoxylin/eosin (HE) staining ofthe remaining mass indicated that the single reovirus injection resultsin the complete destruction of the tumor cells as compared to controltumors (data not shown). To determine if the observed lysis of the tumorcells was due to viral replication and to determine if there was spreadof reovirus proteins beyond the tumor mass, immunofluorescencemicroscopy was conducted. Using antibodies directed against totalreovirus proteins and paraffin embedded thin sections of the remainingtumor mass it was determined that reovirus replication is restricted tothe MDA-MB-468 tumor cells and does not extend into the surroundingskeletal muscle.

[0174] Example 4: Reovirus can Replicate in Primary Breast Specimens

[0175] To ensure that the oncolytic effect of reovirus is not due to aninnate characteristic of a passaged cell line, primary samples of humanbreast cancers were obtained to assess for reovirus infectability.Biopsy breast tumor samples were sterilized by immersion in 95% ethanolfollowed by several washes of sterile PBS. The sample was then slicedinto small sections and placed in a 24 well plate containing DMEM with10% FCS. Reovirus was added (1×10⁸PFUs). At various times postinfection, samples were washed in sterile PBS and then fixed informalin. Samples were then embedded in paraffin and sectioned for usein immunohistochemical analysis using antibodies directed against totalreovirus proteins. The results (not shown) clearly showed reovirusstaining in those tumors challenged, demonstrating viral replication inthese primary samples.

[0176] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

What is claimed is:
 1. A method of determining susceptibility of a cellto reovirus infection by measuring constitutive ras-MAP signaling insaid cell, wherein the presence of said constitutive signaling indicatessusceptibility to infection by reovirus.
 2. The method of claim 1wherein the ras-MAP signaling is measured by determining the state ofphosphorylation of MAP kinase.
 3. The method of claim 2 wherein thestate of MAP kinase phosphorylation is determined using an antibodyspecific for phosphorylated MAP kinase.
 4. The method of claim 1 whereinthe cell is comprised in a biological sample collected from a mammalsuspected of having a proliferative disorder.
 5. The method of claim 4wherein the proliferative disorder is neurofibromatosis.
 6. The methodof claim 4 wherein the proliferative disorder is a solid neoplasm. 7.The method of claim 4 wherein the proliferative disorder is selectedfrom the group consisting of lung cancer, prostate cancer, colorectalcancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer,pancreatic cancer, breast cancer and central and peripheral nervoussystem cancer.
 8. The method of claim 4 wherein the proliferativedisorder is breast cancer.
 9. The method of claim 4 wherein the mammalis selected from the group consisting of dogs, cats, sheep, goats,cattle, horses, pigs, mice, non- human primates, and humans.
 10. Themethod of claim 4 wherein the mammal is human.
 11. A method of treatinga proliferative disorder in a mammal, which disorder is characterized byproliferating cells which exhibit constitutive MAPK phosphorylation,comprising administering to the proliferating cells in said mammal aneffective amount of one or more reoviruses under conditions which resultin substantial lysis of the proliferating cells.
 12. The method of claim11 further comprising a step selected from the group consisting of:administering to the proliferating cells in said mammal an effectiveamount of an immune suppressive agent; removing B-cells or T-cells fromsaid mammal; removing anti-reovirus antibodies from said mammal;removing antibodies from said mammal; administering anti-antireovirusantibodies to said mammal; and suppressing the immune system of themammal.
 13. The method of claim 11, wherein the reovirus is selectedfrom the group consisting of a mammalian reovirus and an avian reovirus.14. The method of claim 13, wherein the reovirus is a mammalianreovirus.
 15. The method of claim 14, wherein the reovirus is a humanreovirus
 16. The method of claim 15, wherein the reovirus is selectedfrom the group consisting of serotype 1 reovirus, serotype 2 reovirusand serotype 3 reovirus.
 17. The method of claim 16, wherein thereovirus is serotype 3 reovirus.
 18. The method of claim 13, wherein thereovirus is an avian reovirus.
 19. The method of claim 11, wherein morethan one type of reovirus is administered.
 20. The method of claim 11,wherein more than one strain of reovirus is administered.
 21. The methodof claim 11, wherein the reovirus is a field isolate.
 22. The method ofclaim 11, wherein the proliferative disorder is a neoplasm.
 23. Themethod of claim 11, wherein the proliferative disorder isneurofibromatosis.
 24. The method of claim 22, wherein the neoplasm is asolid neoplasm.
 25. The method of claim 22, wherein the neoplasm isselected from the group consisting of lung cancer, prostate cancer,colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, livercancer, pancreatic cancer, breast cancer and central and peripheralnervous system cancer.
 26. The method of claim 22, wherein the neoplasmis a central nervous system cancer.
 27. The method of claim 22, whereinthe neoplasm is breast cancer.
 28. The method of claim 22, wherein theneoplasm is a hematopoietic neoplasm.
 29. The method of claim 24,wherein the reovirus is administered by injection into or near the solidneoplasm.
 30. The method of claim 11, wherein the reovirus isadministered intravenously into the mammal.
 31. The method of claim 11,wherein the reovirus is administered intraperitoneally into the mammal.32. The method of claim 11 wherein the mammal is immunocompetent. 33.The method of claim 11 wherein the reovirus is immunoprotected.
 34. Themethod of claim 11 wherein the reovirus is encapsulated in a micelle.35. The method of claim 11, wherein the mammal is a human.
 36. Themethod of claim 11, wherein approximately 1 to 10¹⁵ plaque forming unitsof reovirus/kg body weight are administered.
 37. The method of claim 11,wherein the reovirus is administered in a single dose.
 38. The method ofclaim 11, wherein the reovirus is administered in more than one dose.39. The method of claim 22, wherein the neoplasm is metastatic.
 40. Themethod of claim 11 further comprising the administration of an effectiveamount of a chemotherapeutic agent.
 41. The method of claim 11, whereinthe reovirus is treated with a protease prior to administration.
 42. Themethod of claim 22, wherein the neoplasm is pancreatic cancer.
 43. Themethod of claim 22 further comprising surgical removal of thesubstantially all of the neoplasm and administration of the reovirus tothe surgical site in an amount sufficient to result in substantialoncolysis of any remaining neoplastic cells.
 44. A method of treating aneoplasm in a human, which neoplasm is characterized by proliferatingcells which exhibit constitutive MAPK phosphorylation, comprisingadministering to the neoplasm a reovirus in an amount sufficient toresult in substantial oncolysis of the neoplastic cells.
 45. The methodof claim 44 wherein said reovirus is administered by injection into ornear a solid neoplasm.
 46. The method of claim 44 further comprising astep selected from the group consisting of: administering to theproliferating cells in said mammal an effective amount of an immunesuppressive agent; removing B-cells or T-cells from said mammal;removing anti-reovirus antibodies from said mammal; removing antibodiesfrom said mammal; administering anti-antireovirus antibodies to saidmammal; and suppressing the immune system of the mammal.
 47. A method ofinhibiting metastasis of a neoplasm in a mammal, which neoplasm ischaracterized by proliferating cells which exhibit constitutive MAPKphosphorylation, comprising administering to the mammal a reovirus in anamount sufficient to result in substantial lysis of the neoplasticcells.
 48. The method of claim 47 further comprising a step selectedfrom the group consisting of: administering to the proliferating cellsin said mammal an effective amount of an immune suppressive agent;removing B-cells or T-cells from said mammal; removing anti-reovirusantibodies from said mammal; removing antibodies from said mammal;administering anti-antireovirus antibodies to said mammal; andsuppressing the immune system of the mammal.
 49. A method of treating asuspected neoplasm in a mammal, which neoplasm is characterized byproliferating cells which exhibit constitutive MAPK phosphorylation,comprising surgical removal of substantially all of the neoplasm andadministration of an effective amount of reovirus at or near to thesurgical site resulting in oncolysis of any remaining neoplastic cells.50. The method of claim 49 further comprising a step selected from thegroup consisting of: administering to the proliferating cells in saidmammal an effective amount of an immune suppressive agent; removingB-cells or T-cells from said mammal; removing anti-reovirus antibodiesfrom said mammal; removing antibodies from said mammal; administeringanti- antireovirus antibodies to said mammal; and suppressing the immunesystem of the mammal.